Neuroprotection by a Nuclear Carbonic Anhydrase in C. elegans

线虫核碳酸酐酶的神经保护作用

基本信息

批准号：
8131788
负责人：
Keith Nehrke
金额：
$ 32.8万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8131788
关键词：
Ablation Address Anabolism Animal Model Area Attenuated Automobile Driving Behavioral Assay Bicarbonates Biochemical Biological Assay Biology Biosensor Brain Buffers Caenorhabditis elegans Candidate Disease Gene Carbon Dioxide Catalytic Domain Cell Culture Techniques Cell Death Cell Nucleus Cell Survival Cell physiology Cells Cellular Morphology Cellular Stress Cerebrum Cytoplasm Data Disease Electrolytes Environment Equilibrium Exposure to Fatty acid glycerol esters Functional disorder Gene Expression Regulation Genetic Genetic Models Glucose Goals Homeostasis Human Hypoxia Hypoxia Inducible Factor Immunologic Techniques Impaired cognition Injury Ischemia Label Lesion Life Ligation Mammals Measures Mediating Memory Minerals Modeling Molecular Mus Nematoda Nerve Degeneration Nervous system structure Neurodegenerative Disorders Neurons Nuclear Organism Orthologous Gene Output Oxidation-Reduction Oxidative Stress Oxygen Phenotype Physiological Physiological Processes Process Proteins Protons Recombinants Regulation Reporter Respiration Role Signal Transduction Slice Source Staining method Stains Stress Stroke Techniques Testing Tissues Transgenes Transgenic Organisms Translating Tumor Markers Variant Work bone carbonate dehydratase cellular imaging computerized data processing deprivation design improved loss of function man middle cerebral artery mouse model mutant neuron loss neuroprotection novel overexpression pH Homeostasis preconditioning promoter protective effect public health relevance research study response stress management sugar synaptic function therapeutic target tool

项目摘要

DESCRIPTION (provided by applicant): Carbonic anhydrase (CA) catalyzes the conversion of CO2 to a proton (H+) and bicarbonate (HCO3-) and is involved in many physiologic and pathophysiologic processes. Our preliminary data demonstrate that the nematode C. elegans expresses a CA that selectively localizes to the cell nucleus, is induced by hypoxia, and when lost results in neurodegeneration (ie dysfunction followed by cell death). This is the first example of a classic a-CA that is targeted to the nucleus in any organism. Our central hypothesis is that nuclear CA (NCA) activity protects neurons from hypoxic stress by buffering nuclear pH. We have developed a set of tools that will allow us to study neurodegeneration in a stain that is deficient in NCA and to assess whether its activity is significant for physiologic responses to hypoxia. We will also study signaling processes that are relevant to NCA expression. Finally, using novel genetically-encoded biosensors we will test whether nuclear CA can buffer pH in neuronal nuclei and what effect this has on nuclear redox status (or oxidative stress, which follows hypoxia). All of these approaches will utilize integrative physiologic techniques in live worms. These experiments are geared toward defining the mechanism whereby nuclear CA promotes cell function and viability. A second goal of this proposal is to test whether nuclear CA activity protects mammalian neurons during ischemia. Preliminary evidence demonstrates that transgenic expression of the nematode nuclear CA in mammalian cortical neurons is protective during oxygen-glucose deprivation and hypoxia. In addition to pursuing these studies, the cortical cell culture model will be used as a tissue source for a biochemical approach to identifying a predicted endogenous mammalian NCA. There are currently two candidate genes that are induced by hypoxia and cell stress, respectively, that will be examined using immunologic techniques and recombinant expression assays. In addition, a mouse middle cerebral artery ligation stroke model will be used to determine whether endogenous NCA activity is regulated by hypoxia in an intact brain, with a focus on our two candidate gene products. We hypothesize that worms require a dedicated nuclear CA because of their constant exposure to the environment, but that mammals express or target a CA to the nucleus only under stress conditions. The experiments proposed in this application are focused on defining a novel neuroprotective mechanism that involves pH and electrolyte homeostasis in the cell nucleus mediated by nuclear CA, and as such bridges two relatively diverse, but extremely significant, areas of biology. PUBLIC HEALTH RELEVANCE: We have identified an a-carbonic anhydrase in the genetic model organism C. elegans that regulates neuronal cell death decisions that occur in response to stress, and in particular hypoxia, by buffering the pH of the nucleus. Nuclear pH regulation is a novel mechanism for protecting neurons against ischemia and/or oxidative stress such as occurs in neurodegenerative disease. This application is focused on understanding the mechanism behind nuclear CAs protective effects in nematodes and translating these findings to mammals as a way of targeting cognitive impairment.

描述（由申请人提供）：碳酸酐酶（CA）催化二氧化碳转化为质子（H+）和碳酸氢盐（HCO3-），并参与了许多生理和病理生理过程。我们的初步数据表明，线虫秀丽隐杆线虫表达了一个Ca，该CA有选择地定位于细胞核，是由缺氧诱导的，而当神经变性造成的结果丢失时（即功能障碍，然后是细胞死亡）。这是经典的A-CA的第一个例子，该例子针对任何生物体中的核。我们的中心假设是核CA（NCA）活性通过缓冲核pH来保护神经元免受低氧应激。我们已经开发了一组工具，使我们能够以缺乏NCA的污渍来研究神经退行性，并评估其活性是否对缺氧的生理反应很重要。我们还将研究与NCA表达相关的信号传导过程。最后，使用新型的遗传编码生物传感器，我们将测试核CA是否可以缓冲神经元核中的pH值以及这对核氧化还原状态（或氧化应激）有何影响，而氧化应激，后者是缺氧。所有这些方法都将利用活蠕虫中的综合生理技术。这些实验旨在定义核CA促进细胞功能和活力的机制。该提案的第二个目标是测试核CA活性是否保护缺血期间的哺乳动物神经元。初步证据表明，在氧气 - 葡萄糖剥夺和缺氧期间，哺乳动物皮质神经元中线虫核CA的转基因表达具有保护性。除了进行这些研究外，皮质细胞培养模型还将用作生化方法的组织来源，以鉴定预测的内源性哺乳动物NCA。当前有两个候选基因分别由缺氧和细胞应激诱导，这些基因将使用免疫学技术和重组表达测定法进行检查。此外，将使用小鼠中大脑连接中风模型来确定内源性NCA活性是否受到完整大脑中缺氧的调节，重点是我们的两个候选基因产物。我们假设蠕虫需要专用的核CA，因为它们不断暴露于环境，但是哺乳动物仅在应力条件下将CA表达或靶向CA。本应用中提出的实验的重点是定义一种新型的神经保护机制，该机制涉及由核CA介导的细胞核中的pH和电解质稳态，以及此类桥接两个相对多样但非常重要的生物学领域。公共卫生相关性：我们已经在遗传模型有机体中鉴定了一种A-谐波赤霉酶C.秀丽隐杆线虫，该蛋白隐杆线虫通过缓冲核的pH来缓冲核的pH来调节响应压力，尤其是缺氧而发生的神经元细胞死亡决策。核pH调节是保护神经元免受缺血和/或氧化应激（例如神经退行性疾病中发生）的新型机制。该应用的重点是理解线虫中核壳体保护作用背后的机制，并将这些发现转化为哺乳动物，以此作为靶向认知障碍的一种方式。